Rheumatoid arthritis (RA) is a chronic inflammation of the synovium that causes bone deformity via the progressive destruction of articular cartilage resulting in bone erosion, thus leading to a great decrease in the quality of life of persons suffering from the disease. In spite of extensive studies, the cause of RA remains unclear. Hence, it is important to provide an opportunity for early treatment of RA by rapid and accurate diagnosis before irreversible destruction of joints occurs in order to prevent bone deformity and destruction, thereby improving the prognosis and the quality of life of the patient.
Currently, the diagnosis of RA primarily depends on clinical symptoms, but is, for the most cases, carried out only after the significant progression of joint destruction. Although rheumatoid factor (RF) is adapted as a serological parameter for RA diagnosis in the international classification criteria established by the American College of Rheumotology (ACR), RF is of poor sensitivity because as high as 20% of RA patients are found to be RF negative throughout the progression of RA. Further, RF is low in specificity and it is detected in patients suffering from other rheumatoid disorders, chronic inflammation, or malignant tumors, and is even detected in some healthy persons.
Extensive research has been directed towards the development of other diagnostic markers for RA, resulting in finding the facilitation of citrullination of proteins and the presence of anti-citrullinated protein antibody (ACPA), an autoantibody against citrullinated proteins, in rheumatoid joints. Hence, ACPA is now used as a diagnostic marker for RA.
Citrullination is the conversion of the amino acid arginine in a protein into the amino acid citrulline by deimination as a result of the activity of pepdidylarginine deiminase (PAD) during post-translational modification. At a neutral pH, arginine is positively charged whereas citrulline is uncharged, so that conversion from arginine to citrulline may have crucial influence on the structure and function of the protein. For example, citrullination increases the hydrophobicity of the protein, leading to changes in protein folding, and even in pathological conditions.
The diagnosis of RA utilizing ACPA as a diagnostic marker is implemented by detecting ACPA in a specimen of interest. In detail, a citrullinated protein, for example, natural or recombinant filaggrin is attached onto micro-well plates to which specimen samples are then allocated to induce an antigen-antibody reaction which may lead to the detection of the autoantibodies in the specimens, as analyzed by ELISA.
Based on the finding of a study that highly valuates the advantage of a beta-sheet protein for antigen-antibody reactions over a linear protein structure, a cyclic citrullinated peptide derived from the filaggrin peptide rather than filaggrin itself has recently been used to detect these autoantibodies by ELISA (anti-CCP assay). Found to be superior in diagnostic performance to the conventional anti-citrullinated protein antibody assay using linear peptides, the anti-CCP assay is now predominantly employed for clinical diagnosis of RA. In practice, anti-CCP assay kits have been developed by many manufacturers and are now commercially available. In principle, the anti-CCP assay is based on ELISA wherein, for example, a recombinant human cyclic citrullinated filaggrin peptide, usually produced using a genetic engineering technique, is attached to micro-well plates to which a specimen sample is then allocated to each well to induce an antigen-antibody reaction, followed by color development with an enzyme-conjugated secondary antibody and then by reading absorbance at a certain wavelength (e.g., 400˜600 nm) to determine the presence of the anti-CCP antibody in the specimen.
The anti-CCP assay is based on the premise that a small amount of citrullinated proteins relevant to RA might be present in a specimen from a RA patient, with a large amount of autoandibodies amplified against the citrullinated proteins therein. In practice, however, there are lots of autoantigens and autoantibodies irrelevant to RA in the blood of RA patients, and not all autoantigen candidates are citrullinated (FIG. 1). Further, the secondary antibodies used in the conventional anti-CCP assay kits are usually anti-mouse antibodies specific for mouse antigens, but can recognize non-specific antigens if any are in the specimen sample, giving rise to an increase in false positive errors (FIG. 2).